Plant biomass saccharifying enzyme composition

Abstract

To provide an enzyme formulation that is effective for lowering viscosity in the saccharification process of a plant biomass, and a method for producing the same.SOLUTION: An enzyme composition contains the following (A), (B) and (C): (A) a cellulase; (B) homogalacturonan-degrading enzymes containing a polygalacturonase and a pectin lyase; and (C) one or more selected from an arabinofuranosidase and a galactanase.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an enzyme composition for saccharifying plant biomass. [Background technology]

[0002] Biorefinery technology, which produces sugar and value-added products from renewable plant biomass raw materials, is expected to be a method for building a sustainable society. Among biomass raw materials, agricultural waste containing starch and cellulose can be decomposed to produce a large amount of glucose, the sugar most commonly used as a fermentation raw material, so many technological developments have been carried out to recover sugar from these biomass raw materials. In addition to starch and cellulose, it is known that plant biomass raw materials also contain hemicellulose, pectin, and lignin.

[0003] Pectin is found in large quantities in the primary cell walls and the intercellular spaces of plants, and is a complex polysaccharide mainly composed of three domains: homogalacturonan (HG), rhamnogalacturonan I (RG-I), and rhamnogalacturonan II (RG-II). HG is a polymer mainly composed of polygalacturonic acid in which D-galacturonic acid, in which some of the carboxyl groups at the 6-position have been methylated and some of the hydroxyl groups at the 2-position and 3-position have been acetylated, is linked to α-1,4-linked D-galacturonic acid. Rhamnogalacturonan I (RG-I) is a polymer with a main chain of repeating disaccharides of rhamnose and galacturonic acid, and with galactan side chains consisting of linked galactose residues, arabinan side chains consisting of linked arabinose residues, and arabinogalactan side chains consisting of arabinose and galactose residues. Rhamnogalacturonan II (RG-II) has the most complex structure of all pectins. It has a main chain of α-1,4-linked polygalacturonic acid, and side chains consisting of about 30 kinds of sugars, such as L-rhamnose, D- or L-galactose, L-arabinose, D-galacturonic acid, D-glucuronic acid, D-apiose, L-fucose, 2-O-methyl-L-fucose, 2-O-methyl-D-xylose, aceric acid (3-O-carboxy-5-deoxy-L-xylose), 2-keto-3-deoxy-D-manno-2-octulosonic acid, and 3-deoxy-D-lyxo-2-heptulonic acid, are attached to the O-2 or O-3 position of the galacturonic acid.

[0004] Pectin does not contain glucose, and is therefore not thought to affect the amount of glucose that can be recovered from plant residues. However, the high viscosity of pectin poses a major challenge to the efficiency of sugar recovery from the sugar liquid obtained by decomposing plant biomass and to the sugar production process. The enzymatic saccharification process of pectin-containing plant biomass requires amylase and amyloglucosidase to convert starch to glucose, and cellulase to convert cellulose to glucose, but in addition, in order to reduce the viscosity of the sugar solution, pectinase is used in combination with cellulase. For example, the addition of polygalacturonase (Patent Document 1) and arabinan endo-1,5-α-L-arabinosidase (Patent Document 2) are known.

[0005] However, the viscosity-reducing effect is not necessarily sufficient, and commercially available pectinase preparations contain many other enzymes in addition to the enzyme involved in viscosity reduction, resulting in a large amount being required, which is not practical from a cost perspective. Therefore, there is a demand for an enzyme preparation that can reduce the viscosity of the sugar solution at low cost when saccharifying pectin-containing plant biomass. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Special Publication No. 59-37953 [Patent Document 2] International Patent Publication No. 2015 / 097017 Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention relates to providing an enzyme preparation that is effective for reducing the viscosity during saccharification of plant biomass, and a method for producing the same. [Means for solving the problem]

[0008] The inventors have discovered that an enzyme composition comprising a combination of cellulase and a specific pectinase can effectively suppress the increase in viscosity of a slurry during saccharification of pectin-containing plant biomass, and that the enzyme composition can be easily produced using recombinant filamentous fungi.

[0009] That is, the present invention relates to the following 1) to 8). 1) An enzyme composition comprising the following (A), (B) and (C): (A) cellulase, (B) homogalacturonan degrading enzymes including polygalacturonase and pectin lyase; (C) one or more enzymes selected from arabinofuranosidase and galactanase. 2) A method for saccharifying plant biomass, comprising a step of contacting the enzyme composition of 1) with plant biomass. 3) A method for producing sugar from plant biomass, comprising a step of contacting the enzyme composition of 1) with plant biomass. 4) A method for producing ethanol from plant biomass, comprising a step of contacting the enzyme composition of 1) with plant biomass. 5) A recombinant filamentous fungus into which one or more genes selected from (b1) a polygalacturonase gene, (b2) a pectin lyase gene, and (c) an arabinofuranosidase gene and a galactanase gene have been introduced. 6) A method for producing the enzyme composition of 1), comprising culturing the recombinant filamentous fungus of 5). 7) A plant biomass viscosity reducer comprising the enzyme composition of 1) as an active ingredient. 8) A method for reducing the viscosity of plant biomass, comprising a step of contacting the enzyme composition of 1) with plant biomass. Effect of the Invention

[0010] According to the present invention, it is possible to provide an inexpensive saccharification enzyme composition that suppresses the increase in viscosity of the slurry during the saccharification process of plant biomass containing pectic substances, thereby enabling efficient saccharification of plant biomass. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] (1. Enzyme Composition) The enzyme composition of the present invention comprises the following (A), (B), and (C): (A) cellulase, (B) homogalacturonan degrading enzymes including polygalacturonase and pectin lyase; (C) one or more enzymes selected from arabinofuranosidase and galactanase, It includes.

[0012] (A) Cellulase is a general term for enzymes that degrade cellulose, and includes endoglucanases (EC 3.2.1.4), which cleave cellulose from the inside of the molecule, exoglucanases (cellobiohydrolases, EC 3.2.1.91), which degrade cellulose from the reducing or non-reducing end and release cellobiose, and β-glucosidases (EC 3.2.1.21). In the present invention, cellulase may belong to any of these classes, and multiple types may be used in combination.

[0013] Known sources of such cellulases include Bacillus subtilis, Aspergillus niger, Irpex lacteus, Humicura insorens, Trichoderma viride, and Trichoderma reesei, but in the present invention, it is preferable to use cellulases derived from the genus Trichoderma.

[0014] In the present invention, the cellulase may be in the form of a culture obtained by culturing the above-mentioned cellulase-producing bacteria in a solid or liquid state according to a conventional method, a concentrate obtained by purifying the culture, or a crude enzyme powder obtained by drying the culture, etc. Also, commercially available cellulase preparations, such as Celluclast (NOVOZYME), Meicelase (Meiji Seika Pharma Co., Ltd.), Sumiteam C, Sumiteam AC (Shin Nippon Chemical Industry Co., Ltd.), etc., can be used.

[0015] From the standpoint of saccharification of biomass and suppression of high viscosity of the slurry, cellulase is blended, for example, so that the cellulase activity of the enzyme composition is 2.0 U or more, preferably 2.2 U or more, more preferably 2.4 U or more, and 5.0 U or less, preferably 4.0 U or less, more preferably 3.6 U or less, per 1 mg of protein in the enzyme composition, or 2.0 to 5.0 U, preferably 2.2 to 4.0 U, more preferably 2.4 to 3.6 U. Here, cellulase activity is a value measured by reacting cellulase with filter paper as a substrate at pH 4.8 and 50°C, and quantifying the reducing sugars that are the enzyme reaction products using a colorimetric method, and one unit of enzyme (1 U) means the amount of enzyme that produces 1 μmol of reducing sugars equivalent to glucose per minute.

[0016] The homogalacturonan-degrading enzyme (B) is an enzyme that degrades the homogalacturonan (HG) domain in pectin, and in the present invention includes at least polygalacturonase and pectin lyase. Polygalacturonase is an enzyme (EC 3.2.1.15) that hydrolyzes the α-1,4-glycosidic bond of D-galacturonic acid, which constitutes pectic acid. Pectin lyase is an enzyme (EC 4.2.2.10) that catalyzes the conversion of pectin into oligosaccharides with an unsaturated bond between the C-4 and C-5 terminal residues.

[0017] The polygalacturonase and pectin lyase of the present invention may be derived from the genera Aspergillus, Penicillium, Rhizopus, etc. The polygalacturonase and pectin lyase may be in the form of a culture obtained by solid culture or liquid culture of a microorganism having polygalacturonase-producing ability or pectin lyase-producing ability, respectively, according to a conventional method, a concentrate obtained by purifying the culture, or a crude enzyme powder obtained by drying the culture. Commercially available polygalacturonase preparations and pectin lyase preparations, such as Sumiteam AP2 (Shin Nippon Chemical Industry Co., Ltd.), Sucrase N (Mitsubishi Chemical), and polygalacturonase solution from Aspergillus niger (Sigma, P4716), may also be used.

[0018] Polygalacturonase is preferably formulated so that the polygalacturonase activity of the enzyme composition is, for example, 30 U or more per 1 mg of protein in the enzyme composition, specifically, 30 U or more, preferably 60 U or more, more preferably 90 U or more, more preferably 150 U or more, and 1200 U or less, preferably 600 U or less, more preferably 450 U or less, or preferably 30 to 1200 U, more preferably 60 to 600 U, more preferably 90 to 450 U, more preferably 150 to 450 U. Here, polygalacturonase activity is a value measured by reacting polygalacturonase with a polygalacturonic acid solution as a substrate at pH 4.0 and 50°C, and quantifying the reducing sugars that are the enzyme reaction products by colorimetry, and 1 unit of enzyme (1 U) means the amount of enzyme that produces 1 μmol of reducing sugars equivalent to D-galacturonic acid per minute.

[0019] Pectin lyase is preferably formulated so that the pectin lyase activity of the enzyme composition is, for example, 1 U or more per 1 mg of protein in the enzyme composition, specifically, 1 U or more, preferably 1.5 U or more, more preferably 1.7 U or more, and 27 U or less, preferably 16 U or less, more preferably 10 U or less, or preferably 1 to 27 U, more preferably 1.5 to 16 U, more preferably 1.7 to 10 U. Here, pectin lyase activity was measured by reacting pectin lyase with a polygalacturonic acid solution as a substrate at pH 4.0 and 50°C, and converting the enzymatic reaction product, unsaturated oligogalacturonide, to unsaturated digalacturonide with a molar extinction coefficient of 4600M -1 cm -1 One unit (1U) of enzyme means the amount of enzyme required to produce 1 μmol of unsaturated oligogalacturonide per minute.

[0020] (c) Arabinofuranosidase is an enzyme that hydrolyzes arabinose side chains from the non-reducing end and is classified as EC 3.2.1.55. Galactanases are hydrolases that hydrolyze the glycosidic bonds of galactan and are classified into EC 3.2.1.89, EC 3.2.1.164, and EC 3.2.1.181. Rhamnogalacturonan I (RG-I) in pectin is a polymer having a main chain of a repeating disaccharide structure of rhamnose and galacturonic acid, and having a galactan side chain of linked galactose residues, an arabinan side chain of linked arabinose residues, and an arabinogalactan side chain composed of arabinose residues and galactose residues. Arabinofuranosidase and galactanase contribute to the degradation of the side chain of the rhamnogalacturonan I (RG-I) structure in pectin. In this sense, in the present invention, the arabinofuranosidase and galactanase are sometimes referred to as pectinase.

[0021] As the arabinofuranosidase and galactanase of the present invention, those derived from the genus Aspergillus, Penicillium, Rhizopus, etc. can be used. The arabinofuranosidase and galactanase can be in the form of a culture obtained by solid culture or liquid culture of a microorganism having arabinofuranosidase-producing ability or galactanase-producing ability, respectively, according to a conventional method, a concentrate obtained by purifying the culture, or a crude enzyme powder obtained by drying the culture, etc. In addition, commercially available arabinofuranosidase preparations and galactanase preparations, such as α-L-arabinofuranosidase (Aspergillus niger) (Megazyme, E-AFASE), α-L-arabinofuranosidase (Aspergillus nidulans) (Megazyme, E-ABFAN), endo β-1,4-galactanase (Aspergillus niger) (Megazyme, E-EGALN), endo β-1,4-galactanase (Cellvibrio japonicus) (Megazyme, E-GALCJ), and endo β-1,4-galactanase (Clostridium thermocellum) (Megazyme, E-GALCT), can also be used.

[0022] Arabinofuranosidase is preferably formulated so that the arabinofuranosidase activity of the enzyme composition is, for example, 0.1 U or more per 1 mg of protein in the enzyme composition, specifically, 0.1 U or more, preferably 1 U or more, more preferably 3 U or more, and 50 U or less, preferably 25 U or less, more preferably 15 U or less, or preferably 0.1 to 50 U, more preferably 1 to 25 U, more preferably 3 to 15 U. Here, arabinofuranosidase activity is measured by reacting arabinofuranosidase with a 4-nitrophenyl-α-L-arabinofuranoside solution as a substrate at pH 4.0 and 50°C, and quantifying the enzyme reaction product, 4-nitrophenol, by colorimetry. One unit of enzyme (1 U) means the amount of enzyme that produces 1 μmol of 4-nitrophenol per minute.

[0023] Galactanase is preferably formulated, for example, so that the galactanase activity of the enzyme composition is 3 U or more per 1 mg of protein in the enzyme composition, specifically, 3 U or more, preferably 5 U or more, more preferably 20 U or more, and 250 U or less, preferably 100 U or less, more preferably 80 U or less, or preferably 3 to 250 U, more preferably 5 to 100 U, more preferably 20 to 80 U. Here, galactanase activity is measured by reacting galactan solution as a substrate with galactanase at pH 4.0 and 50°C, and quantifying the reducing sugars that are the enzyme reaction products by colorimetry, and 1 unit of enzyme (1 U) means the amount of enzyme that produces 1 μmol of reducing sugar equivalent to galactose per minute.

[0024] The enzyme composition of the present invention can be formulated by mixing the above-mentioned three types of enzymes (A), (B), and (C) and, if necessary, various carriers, excipients, and other additives that have been conventionally used in enzyme preparations of this type. The dosage form is a tablet, powder, granule, liquid, etc., and can be formulated by a conventional method.

[0025] In the enzyme composition of the present invention, the content of (B) homogalacturonan degrading enzyme and (C) one or more selected from arabinofuranosidase and galactanase ((B)+(C)) in the protein of the enzyme composition is preferably 1% by mass or more, more preferably 5% by mass or more, more preferably 10% by mass or more, and preferably 50% by mass or less, more preferably 45% by mass or less, more preferably 40% by mass or less. Also, it is preferably 1% by mass to 50% by mass, more preferably 5% by mass to 45% by mass, more preferably 10% by mass to 40% by mass. In addition, in the homogalacturonan-degrading enzyme (B), the ratio of polygalacturonase to pectin lyase used is not particularly limited, and they can be mixed in any ratio.

[0026] The enzyme composition of the present invention may also contain, as appropriate, enzymes other than (A) to (C), such as amylase, glucoamylase, β-glucosidase, glucanase, hemicellulase, xylanase, xylosidase, protease, and lipase.

[0027] The enzyme composition of the present invention is an enzyme composition (plant biomass saccharification agent) for saccharifying plant biomass, preferably pectin-containing plant biomass, and more specifically, is an enzyme composition that reduces the viscosity of saccharified plant biomass compared to when the saccharification is performed using cellulase alone. Biomass to be treated with the enzyme composition includes, for example, cellulose- and pectin-containing materials, such as cassava, sweet potato, potato, and other tubers or their residues, corn, corn hulls, apple residues, citrus peels, etc., and preferably cassava or cassava residues.

[0028] (2. Production of Enzyme Compositions by Recombinant Filamentous Fungi) The enzyme composition of the present invention can also be produced by culturing a recombinant filamentous fungus into which one or more genes selected from (b1) a polygalacturonase gene, (b2) a pectin lyase gene, and (c) an arabinofuranosidase gene and a galactanase gene have been introduced.

[0029] (b1) Examples of the polygalacturonase gene include the PgaB gene, the PgaI gene, and the PgaII gene. Such a polygalacturonase gene is preferably derived from a filamentous fungus, and suitable examples thereof include those derived from microorganisms of the genus Aspergillus (e.g., Aspergillus niger, Aspergillus japonicus, Aspergillus aculeatus), microorganisms of the genus Rhizopus (e.g., Rhizopus oryzae, Rhizopus delemar), microorganisms of the genus Talaromyces (e.g., Talaromyces cellulolyticus), microorganisms of the genus Penicillium (e.g., Penicillium roqueforti), and the like. Among these, the PgaB gene, the PgaI gene, and the PgaII gene of Aspergillus niger are more preferred.

[0030] The PgaB gene of Aspergillus niger has been deposited in the NCBI public database under the accession number XM_025597756 (SEQ ID NO: 1). The PgaI gene of Aspergillus niger has been deposited in the NCBI public database under the accession number XM_025596768 (SEQ ID NO: 3). The PgaII gene of Aspergillus niger has been deposited in the NCBI public database under the accession number XM_025602343.1 (SEQ ID NO: 5).

[0031] (b2) Examples of pectin lyase genes include the PelD gene and the PelF gene. Such a pectin lyase gene is preferably derived from a filamentous fungus, and examples thereof include microorganisms of the genus Aspergillus (e.g., Aspergillus niger, Aspergillus japonicus, Aspergillus ludhuensis, Aspergillus aculeatus), microorganisms of the genus Penicillium (e.g., Penicillium digitatum, Penicillium solitum), and microorganisms of the genus Talaromyces (e.g., Talaromyces rugulosus, Talaromyces cellulolyticus). Among these, the PelD gene and the PelF gene of Aspergillus niger are more preferable.

[0032] The PelD gene of Aspergillus niger is registered in the NCBI database under the accession number XM_025604039.1 (SEQ ID NO: 7). The PelF gene of Aspergillus niger is registered in the NCBI database under the accession number: XM_001401024.2 (SEQ ID NO: 9).

[0033] (c) An example of the arabinofuranosidase gene is the Abf gene, and an example of the galactanase gene is the Gal gene. Such arabinofuranosidase gene and galactanase gene are preferably derived from filamentous fungi, for example, microorganisms of the genus Aspergillus (e.g., Aspergillus niger, Aspergillus japonicus, Aspergillus aculeatus), microorganisms of the genus Rhizopus (e.g., Rhizopus oryzae, Rhizopus delemar), microorganisms of the genus Talaromyces (e.g., Talaromyces cellulolyticus), microorganisms of the genus Trichoderma (e.g., Trichoderma reesei), Preferred examples of the fungus include those derived from Penicillium reesei, Penicillium genus microorganisms (e.g., Penicillium roqueforti, Penicillium chrysogenum), and the like. Among these, the Abf gene and Gal gene of Aspergillus niger are more preferable.

[0034] The Abf gene of Aspergillus niger is registered in the NCBI database under the accession number L29005.1 (amino acid sequence AAC41644.1) (SEQ ID NO: 11). The Gal gene of Aspergillus niger is registered in the NCBI database under the accession number AJ305303.1 (amino acid sequence CAC83735.1) (SEQ ID NO: 13).

[0035] Means for introducing one or more genes selected from the (b1) polygalacturonase gene, (b2) pectin lyase gene, and (c) arabinofuranosidase gene and galactanase gene of the present invention into a filamentous fungus include, for example, introducing a vector or a DNA fragment containing a polynucleotide encoding each enzyme gene into a host filamentous fungus (parent strain). Here, the vector containing the polynucleotide encoding each enzyme gene is an expression vector, preferably an expression vector capable of introducing the polynucleotide into a host filamentous fungus and expressing the polynucleotide in the host. The vector preferably contains the polynucleotide and a control region operably linked thereto. The vector may be a vector capable of autonomously replicating and replicating outside a chromosome, such as a plasmid, or may be a vector that is integrated into a chromosome. Here, "operably linked" means that a gene and a regulatory region are linked in such a way that the gene can be expressed under the control of the regulatory region. The procedure for "operably linking" a gene and a regulatory region is well known to those skilled in the art.

[0036] Specific examples of vectors include pBluescript II SK(-) (Stratagene), pUC vectors such as pUC18 / 19 and pUC118 / 119 (Takara Bio), pET vectors (Takara Bio), pGEX vectors (GE Healthcare), pCold vectors (Takara Bio), pHY300PLK (Takara Bio), pUB110 (Mckenzie, T. et al., 1986, Plasmid 15(2):93-103), pBR322 (Takara Bio), pRS403 (Stratagene), pMW218 / 219 (Nippon Gene), pRI vectors such as pRI909 / 910 (Takara Bio), pBI vectors (Clontech), IN3 vectors (Implanta Innovations), pPTR1 / 2 (Takara Bio), pDJB2 (DJBallance et al., 2011). al., Gene, 36, 321-331, 1985), pAB4-1 (van Hartingsveldt W et al., Mol Gen Genet, 206, 71-75, 1987), pLeu4 (MIGRoncero et al., Gene, 84, 335-343, 1989), pPyr225 (CDSkory et al., Mol Genet Genomics, 268, 397-406, 2002), pFG1 (Gruber, F. et al., Curr Genet, 18, 447-451, 1990), and the like.

[0037] Examples of DNA fragments containing a polynucleotide encoding each enzyme gene include, for example, a PCR-amplified DNA fragment and a restriction enzyme-cleaved DNA fragment. Preferably, the DNA fragment may be an expression cassette containing the polynucleotide of the present invention and a control region operably linked thereto.

[0038] The control region contained in the vector or DNA fragment is a sequence for expressing each enzyme gene of the present invention in a host into which the vector or DNA fragment has been introduced, and examples of such control regions include expression regulatory regions such as promoters and terminators, and replication origins. The type of the control region can be appropriately selected depending on the type of host cell into which the vector or DNA fragment is introduced. If necessary, the vector or DNA fragment may further have a selection marker such as an antibiotic resistance gene or an amino acid synthesis-related gene.

[0039] Preferably, the control region contained in the vector or DNA fragment is a control region (so-called strong control region) that is operably linked to the upstream of one or more genes selected from (b1) polygalacturonase gene, (b2) pectin lyase gene, and (c) arabinofuranosidase gene and galactanase gene and has the function of constitutively expressing or highly expressing downstream genes. Examples of the strong control region include the cbh1 promoter, cbh2 promoter, egl1 promoter, xyn1 promoter, xyn2 promoter, and xyn3 promoter of Trichoderma reesei. Further examples of strong regulatory regions include, but are not limited to, regulatory regions of rRNA operons, regulatory regions of genes encoding ribosomal proteins, and the like.

[0040] The target polynucleotide and the control region contained in the vector or DNA fragment may be introduced into the nucleus of the host, or may be introduced into the host genome. Alternatively, the target polynucleotide contained in the vector or DNA fragment may be directly introduced into the host genome and operably linked to a high expression promoter on the genome. A method for introducing a polynucleotide into a genome includes homologous recombination.

[0041] To introduce a vector or DNA fragment into the above-mentioned host cells, a common transformation method such as electroporation, transformation, transfection, conjugation, protoplast, particle gun, or Agrobacterium can be used.

[0042] A recombinant filamentous fungus into which a vector or DNA fragment of interest has been introduced can be selected using a selection marker. For example, when the selection marker is an antibiotic resistance gene, transformed cells into which a vector or DNA fragment of interest has been introduced can be selected by culturing the cells in a medium containing the antibiotic. For example, when the selection marker is an amino acid synthesis-related gene, the gene is introduced into a filamentous fungal strain that requires the amino acid, and then the filamentous fungal strain into which a vector or DNA fragment of interest has been introduced can be selected based on the presence or absence of the amino acid requirement. Alternatively, the introduction of a vector or DNA fragment of interest can be confirmed by examining the DNA sequence of the recombinant strain by PCR or the like.

[0043] By the above-mentioned procedure, a recombinant filamentous fungus can be prepared in which one or more genes selected from (b1) the polygalacturonase gene, (b2) the pectin lyase gene, and (c) the arabinofuranosidase gene and the galactanase gene of the present invention have been introduced into a filamentous fungal strain. The recombinant filamentous fungus of the present invention has the ability to produce cellulase, homogalacturonan degrading enzyme, and arabinofuranosidase and / or galactanase.

[0044] The filamentous fungus used as a host (parent strain) in the present invention is not limited as long as it is a fungus that produces cellulase as a biomass saccharification enzyme and is of a different genus or species from the one or more genes selected from (b1) a polygalacturonase gene, (b2) a pectin lyase gene, and (c) an arabinofuranosidase gene and a galactanase gene to be introduced, and examples of such filamentous fungi include filamentous fungi belonging to the phylum Eumycota and the phylum Oomycota. Specifically, examples of the filamentous fungi include those of the genus Trichoderma, Aspergillus, Penicillium, Neurospora, Fusarium, Chrysosporium, Humicola, Emericella, Hypocrea, Acremonium, Chrysosporium, Myceliophthora, Piromyces, Talaromyces, Thermoascus, and Thielavia, among others, with the genus Trichoderma being preferred.

[0045] Examples of the filamentous fungi of the genus Trichoderma include Trichoderma reesei, Trichoderma longibrachiatum, Trichoderma harzianum, Trichoderma koningii, and Trichoderma viride, among which Trichoderma reesei is preferred, and Trichoderma reesei strain PCD-10 (FERM P-8172) and Trichoderma reesei strain PC-3-7 (ATCC66589) are more preferred.

[0046] The parent filamentous fungus may be a wild-type strain, a strain artificially bred from the wild-type strain, or a mutant strain (mutant) or variant in which a nucleotide sequence in the genome has been substituted, added, deleted or modified.

[0047] Suitable examples of the recombinant filamentous fungus of the present invention include recombinant filamentous fungi obtained by introducing the Aspergillus niger PgaB gene, the Aspergillus niger PelD gene, and the Aspergillus niger Abf gene or the Aspergillus niger Gal gene into Trichoderma reesei strain PC-3-7 (ATCC66589) or a mutant thereof, and specific examples thereof include the strains disclosed in the Examples described below.

[0048] The above-mentioned recombinant filamentous fungus is cultured in the presence of a cellulase inducer, and an enzyme is produced and accumulated in the culture. The enzyme is then collected from the culture, thereby producing an enzyme composition comprising (A) cellulase, (B) homogalacturonan-degrading enzymes including polygalacturonase and pectin lyase, and (C) one or more enzymes selected from arabinofuranosidase and galactanase.

[0049] Here, the "cellulase inducer" is not limited as long as it is a substance that induces cellulase production in cellulase-producing filamentous fungi, and examples thereof include compounds selected from cellulose; sophorose; and cellooligosaccharides such as cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose.

[0050] Here, cellulose includes polymers in which glucose is polymerized through β-1,4-glucoside bonds and derivatives thereof. The degree of polymerization of glucose is not particularly limited. Examples of derivatives include carboxymethylated, aldehyded, or esterified derivatives. Furthermore, cellulose may be a glycoside β-glucoside, a complex with lignin and / or hemicellulose, or a complex with pectin or the like. Cellulose may be crystalline cellulose or non-crystalline cellulose.

[0051] The cellulase inducer can be added in any manner, such as all at once (batch method), divided addition (fed-batch method), or continuous addition (feed method). The amount of cellulase inducer added to the medium may be any amount that can induce the filamentous fungus of the present invention to produce cellulase and pectinase, and varies depending on the addition method. The total amount of the cellulase inducer is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 1% by mass or more, and is preferably 40% by mass or less, more preferably 35% by mass or less, and more preferably 30% by mass or less, based on the medium. The amount is preferably 0.1 to 40% by mass, more preferably 0.5 to 35% by mass, and more preferably 1 to 30% by mass. Among these, the amount added in the case of adding all at once is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 1% by mass or more, and is preferably 16% by mass or less, more preferably 14% by mass or less, more preferably 12% by mass or less. Also, it is preferably 0.1 to 16% by mass, more preferably 0.5 to 14% by mass, more preferably 1 to 12% by mass.

[0052] The medium used in the method of the present invention may be either a synthetic medium or a natural medium, so long as it contains nutrients necessary for the growth of the filamentous fungus of the present invention and the production of cellulase and pectinase, such as a carbon source, a nitrogen source, inorganic salts, vitamins, etc.

[0053] The carbon source may be any carbon source that can be assimilated by the recombinant filamentous fungus of the present invention. Specifically, in addition to the cellulase inducers described above, examples include carbohydrates such as glucose and fructose, alcohols such as ethanol and glycerol, and organic acids such as acetic acid. These can be used alone or in combination. As the carbon source, carbohydrates such as glucose and fructose are desirable, and glucose is more preferable. In this case, the amount of glucose added is preferably 0.1% by mass or more, more preferably 0.5% by mass or more, more preferably 2.5% by mass or more, and preferably 15% by mass or less, more preferably 10% by mass or less, and more preferably 5% by mass or less, based on the medium. Also, it is preferably 0.1 to 15% by mass, more preferably 0.5 to 10% by mass, and more preferably 2.5 to 5% by mass. Also, the amount of cellulase inducer and glucose in the medium is preferably 10:1 to 1:1 in mass ratio, more preferably 4:1 to 2:1.

[0054] Examples of the nitrogen source include ammonia, ammonium salts such as ammonium sulfate, nitrogen compounds such as amines, and natural nitrogen sources such as peptone and soybean hydrolysate.

[0055] Examples of inorganic salts include potassium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, and potassium carbonate.

[0056] Examples of vitamins include biotin, thiamine, etc. Furthermore, substances required for the growth of the recombinant filamentous fungus of the present invention can be added as necessary.

[0057] The culture is preferably carried out under aerobic conditions such as shaking culture or aeration and agitation culture. The culture temperature is preferably 10° C. or higher, more preferably 20° C. or higher, more preferably 25° C. or higher, and preferably 50° C. or lower, more preferably 42° C. or lower, more preferably 35° C. or lower. Also, the culture temperature is preferably 10 to 50° C., more preferably 20 to 42° C., more preferably 25 to 35° C. The pH during the culture is 3 to 9, preferably 4 to 5. The culture time is 10 hours to 10 days, preferably 2 to 7 days.

[0058] After the cultivation is completed, the culture is recovered, and if necessary, the cells are disrupted by ultrasonication or pressure, and solid-liquid separation is performed by filtration, centrifugation, or the like. Then, an enzyme composition containing (A) cellulase, (B) homogalacturonan degrading enzymes including polygalacturonase and pectin lyase, and (C) one or more enzymes selected from arabinofuranosidase and galactanase can be obtained by appropriately combining ultrafiltration, salting out, dialysis, chromatography, and the like. The degree of separation and purification is not particularly limited. The culture supernatant or a crude separation and purification product thereof can also be used as the enzyme composition of the present invention.

[0059] (3. Saccharification of Plant Biomass) By using the enzyme composition of the present invention, plant biomass can be decomposed and saccharified to produce monosaccharides. Specifically, the enzyme composition of the present invention and biomass are brought into contact (coexist) in an aqueous medium, and heated while stirring or shaking, whereby plant biomass can be saccharified to produce monosaccharides. In this case, the viscosity of the saccharified slurry can be reduced by 50% or more, preferably 60% or more, and more preferably 70% or more, compared to the case where only cellulase is used for saccharification. That is, according to the enzyme composition of the present invention, in the saccharification treatment of plant biomass such as cassava, which contains a large amount of pectic substances, it is possible to suppress high viscosity and increase the saccharification efficiency. Therefore, the enzyme composition of the present invention can also be a plant biomass viscosity reducer and can be used to reduce the viscosity of plant biomass. That is, by contacting the enzyme composition of the present invention with plant biomass, the viscosity of the plant biomass can be reduced. The viscosity may be, for example, that measured with a Brookfield viscometer at a solids concentration of 10-20% by mass and at 50°C.

[0060] In the saccharification of plant biomass, the pH and temperature of the reaction solution may be within ranges in which cellulase and pectinase are not inactivated. Generally, when the reaction is carried out at normal pressure, the temperature is in the range of 5 to 95°C and the pH is in the range of 1 to 11. The process of saccharification of plant biomass may be carried out in a batch or continuous manner.

[0061] (4. Ethanol Production) Ethanol can be produced by adding ethanol-fermenting microorganisms to the sugar liquid obtained by saccharifying plant biomass and fermenting it. As ethanol-fermenting microorganisms, yeasts of the genus Saccharomyces and Kluyveromyces, bacteria of the genus Zymomonas, and the like are known, but are not limited thereto. Fermentation is carried out, for example, for 24 to 96 hours at a temperature of 26 to 34°C and at a pH of 3 to 6, preferably around pH 4 to 5. The saccharification process and the fermentation process may be carried out separately, or may be carried out simultaneously as a simultaneous simultaneous fermentation (SSF) process. SSF is carried out by adding yeast and enzymes together, and is carried out, for example, for 24 to 96 hours at a temperature of 26 to 34°C and at a pH of 3 to 6, preferably around pH 4 to 5. When carrying out SSF, a pre-saccharification step at a temperature of more than 50°C may be carried out immediately before fermentation. Further details regarding methods for carrying out saccharification and fermentation are known to those skilled in the art.

[0062] The present invention also includes the following as exemplary embodiments, however, the present invention is not limited to these embodiments. <1> An enzyme composition comprising: (A), (B), and (C) (A) cellulase, (B) homogalacturonan degrading enzymes including polygalacturonase and pectin lyase; (C) one or more enzymes selected from arabinofuranosidase and galactanase. <2> For saccharifying plant biomass, <1> An enzyme composition. <3> The viscosity of saccharified plant biomass is reduced compared to that of saccharified plant biomass using cellulase alone. <2> An enzyme composition. <4> The slurry viscosity of plant biomass is reduced by 50% or more, preferably 60% or more, and more preferably 70% or more, compared to when the plant biomass is saccharified using cellulase alone. <3> An enzyme composition. <5> The cellulase activity is 2.0 U or more, preferably 2.2 U or more, more preferably 2.4 U or more, and 5.0 U or less, preferably 4.0 U or less, more preferably 3.6 U or less, or 2.0 to 5.0 U, preferably 2.2 to 4.0 U, more preferably 2.4 to 3.6 U, per mg of protein in the enzyme composition. <1> ~ <4> Any of the enzyme compositions. <6> The polygalacturonase activity is 30 U or more, preferably 60 U or more, more preferably 90 U or more, more preferably 150 U or more, and 1200 U or less, preferably 600 U or less, more preferably 450 U or less, or preferably 30 to 1200 U, more preferably 60 to 600 U, more preferably 90 to 450 U, more preferably 150 to 450 U, per mg of protein in the enzyme composition. <1> ~ <5> Any of the enzyme compositions. <7> pectin lyase activity is 1 U or more, preferably 1.5 U or more, more preferably 1.7 U or more, and 27 U or less, preferably 16 U or less, more preferably 10 U or less, or preferably 1 to 27 U, more preferably 1.5 to 16 U, more preferably 1.7 to 10 U, per mg of protein in the enzyme composition; <1> ~ <6> Any of the enzyme compositions. <8> The arabinofuranosidase activity is 0.1 U or more, preferably 1 U or more, more preferably 3 U or more, and 50 U or less, preferably 25 U or less, more preferably 15 U or less, or preferably 0.1 to 50 U, more preferably 1 to 25 U, more preferably 3 to 15 U, per mg of protein in the enzyme composition. <1> ~ <7> Any of the enzyme compositions. <9> The galactanase activity is 3 U or more, preferably 5 U or more, more preferably 20 U or more, and 250 U or less, preferably 100 U or less, more preferably 80 U or less, or preferably 3 to 250 U, more preferably 5 to 100 U, more preferably 20 to 80 U, per mg of protein in the enzyme composition. <1> ~ <8> Any of the enzyme compositions. <10> The content ratio of (B) homogalacturonan degrading enzyme and (C) one or more selected from arabinofuranosidase and galactanase ((B)+(C)) per protein in the enzyme composition is preferably 1 or more, more preferably 5 or more, more preferably 10 or more, and is preferably 50 or less, more preferably 45 or less, more preferably 40 or less, or preferably 1 to 50, more preferably 5 to 45, more preferably 10 to 40. <1> ~ <9> Any of the enzyme compositions. <11> <1> ~ <10> A method for saccharifying plant biomass, comprising the step of contacting plant biomass with any one of the enzyme compositions described above. <12> <1> ~ <10> A method for producing sugar from plant biomass, comprising contacting the plant biomass with any one of the enzyme compositions described above. <13> <1> ~ <10> A method for producing ethanol from plant biomass, comprising contacting the plant biomass with any one of the enzyme compositions described above. <14> The plant biomass is a pectin-containing plant biomass; <11> ~ <13> Either way. <15> The plant biomass is cassava or a residue thereof; <11> ~ <13> Either way. <16> <1> ~ <10> The viscosity of plant biomass contacted with any one of the enzyme compositions is reduced compared to when the plant biomass is contacted with cellulase alone. <14> or <15> How to. <17> A recombinant filamentous fungus into which one or more genes selected from (b1) a polygalacturonase gene, (b2) a pectin lyase gene, and (c) an arabinofuranosidase gene and a galactanase gene have been introduced. <18> <17> Cultivating the recombinant filamentous fungus of <1> ~ <10> A method for producing any one of the enzyme compositions described above. <19> (b1) the polygalacturonase gene is the PgaB gene, (b2) the pectin lyase gene is the PelD gene, and (c) the Abf gene or the Gal gene; <17> of recombinant filamentous fungi. <20> (b1) the polygalacturonase gene is the PgaB gene, (b2) the pectin lyase gene is the PelD gene, and (c) the Abf gene or the Gal gene; <18> How to. <21> <1> ~ <2> , <5> ~ <10> 2. A plant biomass viscosity reducing agent comprising any one of the enzyme compositions described above as an active ingredient. <22> <1> ~ <2> , <5> ~ <10> A method for reducing viscosity of plant biomass, comprising contacting the plant biomass with any one of the enzyme compositions described above. <23> <1> ~ <2> , <5> ~ <10> 2. Use of any one of the enzyme compositions for reducing the viscosity of plant biomass. EXAMPLES

[0063] Example 1: Preparation of cellulase Cellulase was obtained by culturing Trichoderma reesei strain E1AB1 (Enzyme and Microbial Technology Volume 82, January 2016, Pages 89-95). The culture was carried out as follows. Enzyme production medium (3% KC Floc W-400G (Nippon Paper Industries), 0.14% (NH 4 ) 2 SO 4 , 1.28% diammonium hydrogen citrate, 0.2% KH 2 PO 4 , 0.03% CaCl 2 2H 2 O, 0.03% MgSO 4 7H 22 × 10 spores of the strain were inoculated into 0.1% Bacto Peptone, 0.05% Bacto Yeast Extract, 0.1% Tween 80, 0.1% trace elements, 50 mM tartrate buffer (pH 4.0; all percentages are w / v%). 5 The bacteria were inoculated at a concentration of 1 / mL and cultured with shaking at 28°C for 5 days. The composition of the trace element is as follows: 6mg H 3 BO 3 , 26mg (NH 4 )6Mo 7 O 24 4H 2 O, 100 mg FeCl 3 6H 2 O, 40 mg CuSO 4 5H 2 O, 8 mg MnCl 2 4H 2 O, 200 mg ZnCl 2 The culture was centrifuged, and the supernatant was filtered to obtain cellulase, which was then used in the following study.

[0064] <Example 2: Preparation of pectinase> (1) cDNA synthesis of Aspergillus niger NBRC105649 strain The A. niger NBRC105649 strain was purchased from the National Institute of Technology and Evaluation, Biotechnology Center. The NBRC105649 strain was grown on a pectin medium (1% Pectin, from citrus (Wako Pure Chemical Industries, Ltd.), 0.14% (NH 4 ) 2 SO 4 , 0.2% KH 2 PO 4 , 0.03% CaCl 2 2H 2 O, 0.03% MgSO 4 7H 2The cells were inoculated into 0, 0.1% Bacto Peptone, 0.05% Bacto Yeast Extract, 0.1% Trace element, and 50 mM tartrate buffer (pH 4.0). The cells were cultured at 28°C for 3 days with shaking, and the cultured cells were harvested using Miracloth (Millipore), frozen in liquid nitrogen, and disrupted using a Multi-Beads Shocker (Yasui Kikai). A metal cone was used as the grinding medium, and the cells were disrupted at 1700 rpm for 30 seconds. RNA was extracted from the disrupted cells using the RNeasy Mini Kit (Qiagen) according to the protocol. The RNA solution was then subjected to the SuperScript III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific) to synthesize cDNA for the NBRC105649 strain.

[0065] (2) Construction of expression vectors for Pga, Pel, Abf, Gal, and Bga1 Fragment (A) was amplified by PCR using the Pichia expression vector pD912 (ATUM) as a template and Fw primer 1 (SEQ ID NO: 15) and Rv primer 1 (SEQ ID NO: 16) shown in Table 1. Fragment (B) was amplified by PCR using the cDNA of the NBRC105649 strain as a template and Fw primer 2 (SEQ ID NO: 17) and Rv primer 2 (SEQ ID NO: 18) shown in Table 1. The resulting DNA fragments (A) and (B) were treated according to the protocol of the In-Fusion HD Cloning Kit (Takara Bio) to construct the ligated plasmid pD912-Pga. Fragment (C) was amplified by PCR using cDNA of the NBRC105649 strain as a template and Fw primer 3 (SEQ ID NO: 19) and Rv primer 3 (SEQ ID NO: 20) shown in Table 1. The resulting DNA fragments (A) and (C) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-Pel was constructed. Fragment (D) was amplified by PCR using cDNA of the NBRC105649 strain as a template and Fw primer 4 (SEQ ID NO: 21) and Rv primer 5 (SEQ ID NO: 22) shown in Table 1. The resulting DNA fragments (A) and (D) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-AnAbf was constructed. Fragment (E) was amplified by PCR using cDNA of the NBRC105649 strain as a template and Fw primer 5 (SEQ ID NO: 23) and Rv primer 5 (SEQ ID NO: 24) shown in Table 1. The resulting DNA fragments (A) and (E) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-AnGal was constructed. Fragment (I) was amplified by PCR using cDNA of the NBRC105649 strain as a template and Fw primer X (SEQ ID NO: 48) and Rv primer X (SEQ ID NO: 49) shown in Table 1. The resulting DNA fragments (A) and (I) were treated according to the protocol of the In-Fusion HD Cloning Kit, and the ligated plasmid pD912-Bga1 was constructed.

[0066] [Table 1]

[0067] (3) Preparation of transformants Each of the target gene expression vectors prepared in (2) was transformed into Pichia pastoris PPS-90102 (ATUM) according to the ATUM protocol.

[0068] (4) Cultivation of transformants P. pastoris was cultured as follows. Each transformant was inoculated into BMD 1% medium (0.2 M potassium phosphate (pH 6.0), 13.4 g / L Yeast Nitrogen Base, 0.4 mg / L biotin, 1.1% glucose) and cultured with shaking at 28°C. When the bacterial cells had grown to a certain amount, methanol was added to induce expression. After 3-5 days, the enzyme expression levels of each culture were confirmed by SDS-PAGE, and each culture was centrifuged and filtered to obtain each culture supernatant. The culture supernatant was concentrated and used as various pectinase (Pga, Pel, Abf, Gal) enzyme solutions.

[0069] Example 3: Protein quantification The protein concentration of the enzyme solution was quantified by the Bradford method, which used the Quick Start Protein Assay (Bio-Rad) and calculated based on a calibration curve using bovine serum albumin as the standard protein.

[0070] Example 4: Measurement of enzyme units (1) Pectate lyase activity Pectate lyase activity was measured by mixing 25 μL of 1% (w / v) polygalacturonic acid (polygalacturonic acid sodium salt, Sigma-Aldrich), 10 μL of 0.5 M sodium acetate buffer (pH 4.0), and 45 μL of milliQ water to prepare 80 μL of substrate solution, which was appropriately diluted with milliQ water and added 20 μL of enzyme solution. The reaction was allowed to proceed at 50°C for 5 minutes, after which 100 μL of 50 mM aqueous hydrochloric acid was added to terminate the enzyme reaction. After stirring and mixing, the absorbance at 235 nm was measured. A blank was prepared by adding aqueous hydrochloric acid to the reaction solution treated without the enzyme solution, and then adding the enzyme solution. The amount of unsaturated oligogalacturonide produced was determined by the molar extinction coefficient of unsaturated digalacturonide, 4600 M -1 cm -1 One unit (1 U) of the enzyme was defined as the amount of enzyme that produces unsaturated oligogalacturonide equivalent to 1 μmol of unsaturated digalacturonide per minute under the above reaction conditions.

[0071] (2) Polygalacturonase activity Polygalacturonase activity was measured by mixing 25 μL of 1% (w / v) polygalacturonic acid (polygalacturonic acid sodium salt, Sigma-Aldrich), 10 μL of 0.5 M sodium acetate buffer (pH 4.0), and 45 μL of milliQ water to prepare 80 μL of substrate solution, adding 20 μL of enzyme solution appropriately diluted with milliQ water, and reacting at 50°C for 5 minutes. After adding 100 μL of 3,5-dinitrosalicylic acid reagent, the color of reducing sugar was developed at 99°C for 10 minutes. After quenching in ice water, the absorbance at 540 nm was measured to determine the amount of reducing sugar produced. A blank was prepared by adding 3,5-dinitrosalicylic acid reagent to the reaction solution treated without adding the enzyme, adding the enzyme solution, and similarly developing the color. One unit of enzyme (1U) was defined as the amount of enzyme that liberates reducing sugars equivalent to 1 μmol of D-galacturonic acid per minute under the above reaction conditions. However, when both polygalacturonase and pectin lyase were present in the enzyme solution, the polygalacturonase activity was calculated by subtracting the pectin lyase unit obtained in (1) from the unit calculated from the amount of reducing sugar.

[0072] (3) Arabinofuranosidase activity Arabinofuranosidase activity was measured by adding 20 μL of enzyme solution appropriately diluted with milliQ water to 80 μL of substrate solution consisting of 20 μL of 5 mM 4-nitrophenyl-α-L-arabinofuranoside (Sigma-Aldrich), 10 μL of 0.5 M sodium acetate buffer (pH 4.0), and 40 μL of milliQ water, reacting at 50°C for 5 minutes, adding 100 μL of 0.4 M sodium carbonate, and measuring the absorbance at 420 nm. A blank was prepared by adding the enzyme solution inactivated at 99°C after adding sodium carbonate solution to the reaction solution treated without the enzyme solution. One unit of enzyme (1U) was defined as the amount of enzyme that produces 1 μmol of 4-nitrophenol per minute under the above reaction conditions.

[0073] (4) Galactanase activity Galactanase activity was measured by adding 20 μL of enzyme solution appropriately diluted with milliQ water to 80 μL of substrate solution, which was a mixture of 25 μL of 1% (w / v) galactan (Galactan (Potato), Megazyme), 10 μL of 0.5 M sodium acetate buffer (pH 4.0), and 45 μL of milliQ water. After reacting at 50 °C for 5 minutes, 100 μL of 3,5-dinitrosalicylic acid reagent was added and the color of reducing sugar was developed at 99 °C for 10 minutes. After quenching in ice water, the absorbance at 540 nm was measured to determine the amount of reducing sugar produced. A blank was prepared by adding 3,5-dinitrosalicylic acid reagent to a reaction solution treated without adding enzyme, adding enzyme solution, and coloring in the same manner. One unit of enzyme (1U) was defined as the amount of enzyme that liberates reducing sugar equivalent to 1 μmol of galactose per minute under the above reaction conditions.

[0074] (5) Cellulase activity The cellulase activity was measured by the microplate-scale filter paper decomposition activity test method reported in MICROPLATE-BASED FILTER PAPER ASSAAY TO MEASURE TOTAL CELLULASE ACTIVITY (BIOTECHNOLOGY AND BIOENGINEERING. 2004 DEC 30;88(7):832―7.DOI:10.1002 / BIT.20286.). 20 μL of appropriately diluted sample was added to 40 μL of citrate buffer (50 mM, pH 4.8) containing filter paper (WHATMAN NO.1, GE HEALTHCARE) cut to a diameter of 7 mm, and the reaction was carried out at 50°C for 1 hour. A sample without filter paper was prepared in the same manner as the blank sample. Then, 120 μL of DNS solution was added to stop the reaction, and the mixture was heated at 95°C for 5 minutes and cooled in ice water for 5 minutes. The obtained solution was mixed with 36 μL of milliQ water and the absorbance of the mixture was measured at 540 nm, and the increase in absorbance was calculated by subtracting the blank. Next, the amount of reducing sugar generated in the reaction solution was calculated in terms of the amount of glucose using a calibration curve created with the stepwise diluted glucose concentration and the absorbance at 540 nm. The same operation was performed with samples of different dilution rates, and the dilution rate of the sample required to generate reducing sugar equivalent to 0.08 mg glucose / 20 μL cellulase solution was determined, and the filter paper decomposition activity (FPU / mL) of the sample before dilution was calculated. Furthermore, the cellulase concentration was measured by the method of Example 4, and the display unit of cellulase activity was converted to FPU / mg-PROTEIN. The activity unit was defined as "1 U" for the enzyme activity that generates reducing sugar equivalent to 1 μmol of glucose in 1 minute.

[0075] Example 5: Viscosity measurement The viscosity was measured using a tuning fork vibration viscometer (SV-10H, manufactured by A&D Co., Ltd.).

[0076] Test example: Saccharification of cassava residue <Test Example 1 - Comparative Example> Cassava residue was weighed out to a dry weight of 4 g in a 100 mL baffled Erlenmeyer flask, and α-amylase was added to a substrate concentration of 0.002 mg protein / g and milli-Q water was added to a substrate concentration of 15 wt%. The baffled flask was then thoroughly mixed, sealed with a cotton plug, covered with aluminum foil, and gelatinized at 90°C for 2 hours (autoclave, TOMY LSX-700). After that, it was cooled to 50°C in a shaker (PRECI PRXY-30-R-3F) set to 50°C, and glucoamylase was added to a substrate concentration of 0.05 mg protein / g, and the mixture was allowed to react at 50°C and 200 rpm for 18 hours. After saccharification, the mixture did not become liquid, and the viscosity could not be measured.

[0077] <Test Example 2 - Comparative Example> The same study as in Test Example 1 was carried out, except that enzyme composition 1 shown in Table 2 was further added to the gelatinized substrate at 0.2 mg-protein / g-substrate. The slurry after saccharification did not become liquid, and the viscosity was not measurable.

[0078] <Test Example 3 - Comparative Example> The same study as in Test Example 1 was carried out, except that enzyme composition 2 shown in Table 2 was further added to the gelatinized substrate at 0.2 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 84 mPa s.

[0079] <Test Example 4 - Comparative Example> The same study as in Test Example 1 was carried out, except that enzyme composition 3 shown in Table 2 was further added to the gelatinized substrate at 0.2 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 79 mPa s.

[0080] <Test Example 5 - Comparative Example> The same study as in Test Example 1 was carried out, except that enzyme composition 4 shown in Table 2 was further added to the gelatinized substrate at 0.2 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 459 mPa s.

[0081] <Test Example 6> The same study as in Test Example 1 was carried out, except that enzyme composition 5 shown in Table 3 was further added to the gelatinized substrate at 0.16 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 26.8 mPa s.

[0082] <Test Example 7> The same study as in Test Example 1 was carried out, except that enzyme composition 6 shown in Table 3 was further added to the gelatinized substrate at 0.16 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 16.6 mPa s.

[0083] <Test Example 8> The same study as in Test Example 1 was carried out, except that enzyme composition 7 shown in Table 3 was further added to the gelatinized substrate at 0.16 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 14.5 mPa s.

[0084] <Test Example 9> The same study as in Test Example 1 was carried out, except that enzyme composition 8 shown in Table 3 was further added to the gelatinized substrate at 0.16 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 17.3 mPa s.

[0085] <Test Example 10> The same study as in Test Example 1 was carried out, except that enzyme composition 9 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 47.3 mPa s.

[0086] <Test Example 11> The same study as in Test Example 1 was carried out, except that enzyme composition 10 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 37.9 mPa s.

[0087] <Test Example 12> The same study as in Test Example 1 was carried out, except that enzyme composition 11 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 19.7 mPa s.

[0088] <Test Example 13> The same study as in Test Example 1 was carried out, except that enzyme composition 12 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 17.3 mPa s.

[0089] <Test Example 14> The same study as in Test Example 1 was carried out, except that enzyme composition 13 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 23.1 mPa s.

[0090] <Test Example 15> The same study as in Test Example 1 was carried out, except that enzyme composition 14 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 23.8 mPa s.

[0091] <Test Example 16> The same study as in Test Example 1 was carried out, except that enzyme composition 15 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 19.9 mPa s.

[0092] <Test Example 17> The same study as in Test Example 1 was carried out, except that enzyme composition 16 shown in Table 4 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 39.0 mPa s.

[0093] <Test Example 18 - Comparative Example> The same study as in Test Example 1 was carried out, except that enzyme composition 17 shown in Table 5 was further added to the gelatinized substrate at 0.14 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 115 mPa s.

[0094] <Test Example 19> The same study as in Test Example 1 was carried out, except that enzyme composition 18 shown in Table 5 was further added to the gelatinized substrate at 0.14 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 71.8 mPa s.

[0095] <Test Example 20> The same study as in Test Example 1 was carried out, except that enzyme composition 19 shown in Table 6 was further added to the gelatinized substrate at 0.13 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 49.1 mPa s. <Test Example 21> The same study as in Test Example 1 was carried out, except that enzyme composition 20 shown in Table 6 was further added to the gelatinized substrate at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 47.5 mPa s.

[0096] <Test Example 22 - Comparative Example> The same study as in Test Example 1 was carried out, except that enzyme composition 21 containing the pectin side-chain degrading enzyme AnBga1 shown in Table 7 was further added to the substrate after gelatinization treatment at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 1440 mPa s. AnBga1 is a β-galactosidase that is an exo-type galactan degrading enzyme.

[0097] <Test Example 23 - Comparative Example> The same study as in Test Example 1 was conducted, except that enzyme composition 22 containing E-ARABCJ (Megazyme), a pectin side-chain degrading enzyme shown in Table 7, was further added to the substrate after gelatinization treatment at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 4,010 mPa s. E-ARABCJ is an endo-type arabinan degrading enzyme.

[0098] <Test Example 24 - Comparative Example> The same study as in Test Example 1 was carried out, except that E-BGLAN (Megazyme) enzyme composition 23, a pectin side-chain degrading enzyme shown in Table 7, was further added to the substrate after gelatinization treatment at 0.15 mg-protein / g-substrate. The viscosity of the slurry after saccharification was 530 mPa s. E-BGLAN is a β-galactosidase, an exo-type galactan degrading enzyme.

[0099] [Table 2]

[0100] [Table 3]

[0101] [Table 4]

[0102] [Table 5]

[0103] [Table 6]

[0104] [Table 7]

[0105] Example 6: Construction of cellulase / pectinase producing strain (1) Construction of Gal expression vector The fragment (A) was amplified by PCR using the plasmid pUC-eg3, in which the region from upstream to downstream of the eg3 gene derived from Trichoderma reesei (SEQ ID NO: 47) was inserted into the HincII restriction enzyme cleavage site of pUC118 (Takara Bio) as a template, and Fw primer 6 (SEQ ID NO: 25) and Rv primer 6 (SEQ ID NO: 26) shown in Table 7. The amplified fragment (B) was amplified by PCR using the genomic DNA of Trichoderma reesei as a template and Fw primer 7 (SEQ ID NO: 27) and Rv primer 7 (SEQ ID NO: 28) shown in Table 7. The obtained DNA fragments (A) and (B) were treated according to the protocol of the In-Fusion HD Cloning Kit (Takara Bio) to construct the plasmid pUC-eg3-pyr4 in which the pyr4 gene was linked downstream of the eg3 gene. Furthermore, fragment (C) was amplified by PCR using pUC-eg3-pyr4 as a template and Fw primer 8 (sequence number 29) and Rv primer 8 (sequence number 30) shown in Table 7, and fragment (D) was amplified by PCR using Trichoderma reesei genomic DNA as a template and Fw primer 9 (sequence number 31) and Rv primer 9 (sequence number 32) shown in Table 7, and these were similarly treated with the In-Fusion HD Cloning Kit to obtain plasmid pUC-Δeg3::Pcbh1-cbh1-pyr4. Next, fragment (E) was amplified by PCR using pUC-Δeg3::Pcbh1-cbh1-pyr4 as a template and Fw primer 10 (sequence number 33) and Rv primer 10 (sequence number 34) shown in Table 7, and fragment (F) was amplified by PCR using plasmid pUC-eg3 as a template and Fw primer 11 (sequence number 35) and Rv primer 11 (sequence number 36) shown in Table 7, and these were similarly treated with the In-Fusion HD Cloning Kit to obtain plasmid pUC-Δeg3::Pcbh1-cbh1-pyr4#1000. Next, fragment (G) amplified by PCR using pUC-Δeg3::Pcbh1-cbh1-pyr4#1000 as a template and Fw primer 7 (sequence number 27) and Rv primer 9 (sequence number 32) shown in Table 7, and fragment (H) amplified by PCR using Trichoderma reesei genomic DNA as a template and Fw primer 12 (sequence number 37) and Rv primer 12 (sequence number 38) shown in Table 8 were similarly treated with the In-Fusion HD Cloning Kit to obtain plasmid pUC-Δeg3::Pcbh1-cbh1-pyr4(re). Fragment (I) was amplified by PCR using pUC-Δeg3::Pcbh1-cbh1-pyr4(re) as a template and Fw primer 13 (sequence number 39) and Rv primer 13 (sequence number 40) shown in Table 7, and fragment (J) was amplified by PCR using Trichoderma reesei genomic DNA as a template and Fw primer 14 (sequence number 41) and Rv primer 14 (sequence number 42) shown in Table 7, and the fragment was similarly treated with the In-Fusion HD Cloning Kit to obtain plasmid pUC-Δeg3::Pxyn1-cbh1-pyr4(re). Then, fragment (K) was amplified by PCR using pUC-Δeg3::Pxyn1-cbh1-pyr4(re) as a template and Fw primer 15 (sequence number 43) and Rv primer 15 (sequence number 44) shown in Table 7, and fragment (L) was amplified by PCR using cDNA of Aspergillus niger NBRC105649 strain as a template and Fw primer 16 (sequence number 45) and Rv primer 16 (sequence number 46) shown in Table 7, and these were similarly treated with the In-Fusion HD Cloning Kit to obtain the GAL2 expression plasmid pUC-Δeg3::Pxyn1-AnGAL-pyr4(re).

[0106] [Table 8]

[0107] (2) Preparation of transformants A strain (Patent Application No. 2021-154749) in which pgaB and pelD were expressed in a uracil-requiring strain of Trichoderma reesei E1AB1 strain was designated SP21, and this strain was transformed with the vector constructed in (1) above. Introduction was performed using the protoplast PEG method. The transformants were grown on a selective medium (2.0% glucose, 1.1 M sorbitol, 2.0% agar, 0.2% KH) containing acetamide as the sole nitrogen source. 2 PO 4 , 0.06% CaCl 2 2H 2 O, 0.06% MgSO 4 7H 2 The selection was performed using 0.06% acetamide and 0.1% Trace element 2 (w / v%). The composition of Trace element 2 is as follows: 0.5g FeSO 4 7H 2 O, 0.2g CoCl 2 , 0.16g MnSO 4 H 2 O, 0.14g ZnSO 4 7H 2 The volume of the culture was adjusted to 100 mL with distilled water. The selected transformants were then stabilized by subculture, and the strains that stably retained the target gene were further selected by colony PCR. The resulting transformant was designated SP21·Gal.

[0108] (3) Enzyme production In the enzyme production medium, 2 × 10 spores of SP21 or SP21·Gal were added. 5 The bacteria were inoculated at a concentration of 100 / mL and cultured at 28°C for 5 days with shaking. The cultures were centrifuged and then filtered to obtain the culture supernatants. The culture supernatants were used as enzyme solutions for the study.

[0109] Test example: Saccharification of cassava residue <Test Example 21> The same study as in Test Example 1 was carried out, except that 0.05 mg-protein / g-substrate of glucoamylase and 0.2 mg-protein / g-substrate of SP21-derived culture supernatant were added to the gelatinized substrate. The viscosity of the slurry after saccharification was 60.0 mPa s.

[0110] <Test Example 22> The same study as in Test Example 1 was performed, except that 0.05 mg-protein / g-substrate of glucoamylase and 0.2 mg / g-protein / g-substrate of SP21·Gal-derived culture supernatant were added to the gelatinized substrate. The viscosity of the slurry after saccharification was 18.0 mPa s.

[0111] <Test Example 23> The enzyme units in the culture supernatants derived from SP21 and SP21·Gal were measured, and the results are shown in Table 9.

[0112] [Table 9]

Claims

1. An enzyme composition comprising: (A), (B), and (C) (A) cellulase, (B) homogalacturonan degrading enzymes including polygalacturonase and pectin lyase; (C) one or more selected from arabinofuranosidase and galactanase.

2. The enzyme composition according to claim 1 for saccharifying plant biomass.

3. The enzyme composition according to claim 2, which reduces the viscosity of saccharified plant biomass compared to when the saccharification is performed using only cellulase.

4. 2. The enzyme composition according to claim 1, having a cellulase activity of 2.0 to 3.5 U per mg of protein in the enzyme composition.

5. 2. The enzyme composition according to claim 1, having a polygalacturonase activity of 30 U to 1200 U per mg of protein in the enzyme composition.

6. 2. The enzyme composition according to claim 1, having a pectin lyase activity of 1 to 27 U per mg of protein in the enzyme composition.

7. 2. The enzyme composition according to claim 1, having an arabinofuranosidase activity of 0.1 to 50 U per mg of protein in the enzyme composition.

8. 2. The enzyme composition according to claim 1, having a galactanase activity of 3 to 250 U per mg of protein in the enzyme composition.

9. The enzyme composition according to claim 1, wherein the proportion of the protein (B) homogalacturonan decomposing enzyme and (C) one or more selected from arabinofuranosidase and galactanase ((B) + (C)) in the enzyme composition is 1% by mass to 50% by mass.

10. A method for saccharifying plant biomass, comprising the step of contacting the enzyme composition of claim 1 with the plant biomass.

11. 10. A method for producing sugars from plant biomass, comprising contacting the plant biomass with the enzyme composition of claim 1.

12. 10. A method for producing ethanol from plant biomass, comprising contacting the enzyme composition of claim 1 with the plant biomass.

13. The method according to any one of claims 10 to 12, wherein the plant biomass is a pectin-containing plant biomass.

14. The method according to any one of claims 10 to 12, wherein the plant biomass is selected from tubers such as cassava, sweet potato, potato, etc. or residues thereof, corn, corn hulls, apple residues, and citrus peels.

15. The method according to any one of claims 10 to 12, wherein the plant biomass is cassava or a residue thereof.

16. 14. The method of claim 13, wherein the viscosity of plant biomass contacted with the enzyme composition of claim 1 is reduced compared to contact with cellulase alone.

17. The method of claim 14, wherein the viscosity of plant biomass contacted with the enzyme composition of claim 1 is reduced compared to contact with cellulase alone.

18. The method of claim 15, wherein the viscosity of plant biomass contacted with the enzyme composition of claim 1 is reduced compared to contact with cellulase alone.

19. A recombinant filamentous fungus into which one or more genes selected from (b1) a polygalacturonase gene, (b2) a pectin lyase gene, and (c) an arabinofuranosidase gene and a galactanase gene have been introduced.

20. A method for producing the enzyme composition according to any one of claims 1 to 9, comprising culturing the recombinant filamentous fungus according to claim 19.

21. 20. The recombinant filamentous fungus according to claim 19, wherein (b1) the polygalacturonase gene is the PgaB gene, (b2) the pectin lyase gene is the PelD gene, and (c) the Abf gene or the Gal gene.

22. The method according to claim 20, wherein (b1) the polygalacturonase gene is the PgaB gene, (b2) the pectin lyase gene is the PelD gene, and (c) the Abf gene or the Gal gene.

23. A plant biomass viscosity reducing agent comprising the enzyme composition according to any one of claims 1 to 2 and 4 to 9 as an active ingredient.

24. A method for reducing the viscosity of plant biomass, comprising a step of contacting the enzyme composition according to any one of claims 1 to 2 and 4 to 9 with plant biomass.